The overall goal of this project is to discover novel antibiotics to combat important drug-resistant pathogens. We are running out of treatment options for pathogens such as S. aureus MRSA, vancomycin-resistant Enterococci (VRE), multidrug-resistant P. aeruginosa, A. baumannii, ESBL and New Delhi metallo-b- lactamase-producing Enterobacteriaceae, and M. tuberculosis. Only 3 novel antibiotics have been introduced in the past thirty years - linezolid, daptomycin, and fidaxomicin. Linezolid and fidaxomicin were discovered in the 60s, but did not appear sufficiently attractive at the time. With this pace of discovery, it is not surprising that resistance is on the rise. It is becoming increasingly apparent that the bottleneck in antibiotic discovery is the lack of good starting compounds. Not a single drug came out of HTS of synthetic compound libraries. Secondary metabolites produced by actinomycetes have been the main source of antibiotics, but this resource was over mined. At the same time, there is a potentially very large untapped source of natural products - previously uncultured bacteria that make up the vast majority of all bacterial species. Slow- growing species that require months to form colonies on a Petri dish are an important component of this majority. We reasoned that slow growers may actually represent dormant forms of bacteria, and will rapidly grow upon reinoculation. The majority of slow growers can indeed be rapidly cultured upon reinoculation, and many of the isolates represent previously unknown species and genera. In Phase I, we developed a method to simultaneously isolate and culture slow growers by placing individual cells in wells of a microtiter plate. Screening 5,000 of these isolates produced 3 new antimicrobial compounds, including Novo23 that acts specifically against M. tuberculosis. The target of Novo23 is the ClpC1 subunit of the essential mycobacterial ClpP protease. Novo23 has low cytotoxicity, favorable tolerability and blood levels in mice. We will examine efficacy of Novo23 in mouse models of tuberculosis. Further development of our three novel antibacterials are a major focus of Phase II. However, we recognize that only a small fraction of leads makes it to a drug. Thus, we will also undertake a large-scale discovery effort to identify additional antibacterials which will enter validation as they become available. Novel compounds will be examined for spectrum, potency, resistance development, stability, mechanism of action, and novelty of structure. Leads that emerge will be tested in mouse models of infection. The end result of Phase II will be three lead compounds showing efficacy in animal models. This will enable subsequent preclinical development towards an IND, clinical studies, and FDA approval of a new therapeutic. We believe this strategy - advancing leads while backing them with a discovery pipeline - greatly increases the chances for the project's success.